Combinations for the treatment of diseases involving angiogenesis

ABSTRACT

Compositions for the inhibition of the formation of new vasculature by angiogenesis are provided comprising the combination of a vasculature damaging agent and an inhibitor of the formation or action of nitric oxide in mammalian systems. There are also provided the use of said combinations in medicaments and kits of said compounds and treatment employing said materials.

This invention relates to a method for treating diseases involvingactive angiogenesis, to compositions useful for the treatment ofdiseases involving angiogenesis and to the use of the compositions inthe preparation of a medicament for the treatment of diseases involvingactive angiogenesis. In one aspect of the invention the method involvesthe administration to a mammal of an inhibitor of nitric oxide incombination with a compound inducing vascular damage.

Formation of new vasculature by angiogenesis is a key pathologicalfeature of several diseases (J Folkman, New England Journal of Medicine333, 1757-1763, 1995). For example, for a solid tumour to grow it mustdevelop its own blood supply upon which it depends critically for theprovision of oxygen and nutrients; if this blood supply is mechanicallyshut off the tumour undergoes necrotic death. Neovascularisation is alsoa clinical feature of skin lesions in psoriasis, of the invasive pannusin the joints of rheumatoid arthritis patients and of atheroscleroticplaques. Retinal neovascularisation is pathological in maculardegeneration and in diabetic retinopathy. In all these diseases reversalof neovascularisation by damaging the newly-formed vascular endotheliumis expected to have a beneficial therapeutic effect.

Certain chemical compounds have been shown to have vascular damagingactivity against the newly formed endothelium of solid tumours. Theseagents include, for example, combretastatin A4 phosphate (Dark et al.,Cancer Research 57, 1829-1834, 1997), combretastain analogues (forexample those described in J Med Chem 41, 3022-32,1998 by Ohsumi etal.), the flavone acetic acids, for example 5,6-dimethylxanthenoneacetic acid (Zwi, Pathology, 26, 161-9, 1994), colchicine (Baguley etal. Eur J Cancer 27, 482-7, 1991). However some tumours are resistant tothese agents.

One characteristic of tumours relatively resistant to vascular damagingagents is their ability to produce large amounts of nitric oxide. Therole of nitric oxide in tumour growth is unclear and there have beenreports of both tumour-stimulating and tumour-inhibiting effects (Chinjeand Stratford, Essays Biochem. 32, 61-72, 1997). It has been suggestedthat the antitumour effects of 5,6-dimethylxanthenone acetic acid aremediated in part by nitric oxide production (Thompsen et al. CancerChemother Pharmacol. 31, 151-5, 1992).

WO-A 9509621 and Br. J Cancer (1999), 77(3), 426-433 disclosecombinations of cytokine releasing anticancer agents (TNF-releasingagents). These relate to ameliorating the effects of pro-inflammatorycytokines. There is no suggestion of synergistic activity from acombination of a vascular damaging agent (many of which have nopro-inflammatory activity) and an NO inhibitor.

We have found that the efficacy of vascular damaging agents can beimproved by combining the treatment with inhibitors of the formation oraction of nitric oxide in a mammalian system.

In particular the efficacy of vascular damaging agents can be improvedby combination with inhibitors of nitric oxide synthases, the enzymesthat produce nitric oxide from arginine. In particular the efficacy ofvascular damaging agents against tumours relatively resistant to theireffects is improved by treatment with a nitric oxide synthase inhibitor.

Accordingly in one aspect of the invention we provide a method oftreatment for a mammal having a disease that involves activeangiogenesis such method comprising the administration of a therapeuticor subtherapeutic amount of a vascular damaging agent together with aninhibitor of nitric oxide synthase in an amount sufficient to augmentthe effect of the vascular damaging agent. The method is useful for thetreatment of diseases such as cancers, especially solid tumours,psoriasis, diabetic retinopathy, macular degeneration, atherosclerosisand rheumatoid arthritis.

The vascular damaging agent and the nitric oxide synthase inhibitor canbe administered together or separately. The method may be used as a soletherapy or in combination with other treatments. For the treatment ofsolid tumours compounds of the invention may be administered incombination with radiotherapy or in combination with other anti-tumoursubstances for example those selected from mitotic inhibitors, forexample vinblastine, paclitaxel and docetaxel, alkylating agents, forexample cisplatin, carboplatin and cyclophosphamide, antimetabolites,for example 5-fluorouracil, cytosine arabinoside and hydroxyurea;intercalating agents for example adriamycin and bleomycin; enzymes, forexample aspariginase; topoisomerase inhibitors for example etoposide,topotecan and irinotecan; thymidylate synthase inhibitors for exampleraltitrexed; biological response modifiers for example interferon;antibodies for example edrecolomab; and anti-hormones for exampletamoxifen. Such combination treatment may involve simultaneous orsequential application of the individual components of the treatment.

The vascular damaging agent and the nitric oxide synthase inhibitor canbe administered by the same route or by different routes. Such routes ofadministration include oral, buccal, nasal, topical, rectal andparenteral administration. Each component of the method, the vasculardamaging agent and the nitric oxide synthase inhibitor may independentlybe administered in a form suitable for the intended route ofadministration and such forms may be prepared in a conventional mannerusing conventional excipients. For example for oral administration thepharmaceutical compositions may take the form of tablets or capsules.For nasal administration or administration by inhalation the compoundsmay be conveniently delivered as a powder or in solution. Topicaladministration may be as an ointment or cream and rectal administrationmay be as a suppository. For parenteral injection (includingintravenous, subcutaneous, intramuscular, intravascular or infusion) thecomposition may take the form of, for example, a sterile solution,suspension or emulsion. The preferred route of administration of eachcomponent will depend on the disease being treated. For solid tumoursthe components may each advantageously be delivered, either together orseparately, as an intravenous infusion.

Vascular damaging agents are compounds which induce selective damage tonewly formed, rather than established, vasculature. Many such compoundsare known and it is considered this invention is generally applicable tosuch agents. Such agents include tubulin-binding agents, for example thecombretastatins and their prodrugs, the colchinols and their prodrugsand (Z)-2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]phenylamine and itsprodrugs, TNF-alpha inducing agents such as the xanthenone acetic acids,for example dimethylxanthenoneacetic acid, and antibodies targeted tothe vasculature.

A wide variety of compounds which inhibit the formation or action ofnitric oxide in mammalian systems can be employed. Specifically nitricoxide synthase inhibitors are those compounds which inhibit any of theforms of nitric oxide synthase. Such agents include derivatives ofarginine, ornithine, lysine and citrulline, S-alkylthioureas andaminoguanidines. Where the nitric oxide synthase inhibitor is aderivative of arginine it may be, for example, an N^(G)-substitutedL-arginine selected from N^(G)-nitro-L-arginine and alkyl estersthereof, N^(G)-methyl-L-arginine and N^(G)-amino-L-arginine. Where thenitric oxide synthase inhibitor is a derivative of ornithine it may be,for example L-N6-(1-iminoethyl)-ornithine. Where the nitric oxidesynthase inhibitor is a derivative of lysine it may be, for exampleL-N6-(1-iminoethyl)-lysine. Where the nitric oxide synthase inhibitor isa derivative of citrulline it may be, for example L-thiocitrulline,L-homothiocitrulline or an S-alkylthiocitrulline such asS-methyl-L-thiocitrulline.

In a further embodiment of the invention there is provided a compositionfor the treatment of diseases involving active angiogenesis. Thecomposition of the invention comprises a vascular damaging agent incombination with a nitric oxide synthase inhibitor where both thevascular damaging agent and the nitric oxide synthase inhibitor are ashereinbefore defined.

Thus for example the composition may contain for example acombretastatin derivative, a colchicine derivative, a colchinolderivative, a xanthenone acetic acid derivative or a vascular targetedantibody, in combination with a nitric oxide synthase inhibitor forexample a derivative of arginine, a derivative of ornithine, aderivative of lysine, a derivative of citrulline, a S-alkylthioureas oran aminoguanidine.

Particular examples of vascular damaging agents that may be present inthe composition include combretastatin A4 and its prodrugs for examplecombretastatin A4 phosphate,(Z)-2-methoxy-5-[2-(3,4,5-trimethoxyphenyl)vinyl]phenylamine and itsprodrugs, N-acetylcolchinol and its prodrugs for exampleN-acetylcolchinol-O-phosphate and 5,6-dimethylxanthenoneacetic acid.

Particular examples of nitric oxide synthase inhibitors which may bepresent in the composition include derivatives of arginine, ornithine,lysine and citrulline, S-alkylthioureas aminoguanidines andaminopyridines. Where the nitric oxide synthase inhibitor is aderivative of arginine it may be, for example, an N^(G)-substitutedL-arginine selected from N^(G)-nitro-L-arginine and alkyl estersthereof, N^(G)-methyl-L-arginine and N^(G)-amino-L-arginine. Where thenitric oxide synthase inhibitor is a derivative of ornithine it may be,for example L-N6-(1-iminoethyl)-ornithine. Where the nitric oxidesynthase inhibitor is a derivative of lysine it may be, for exampleL-N6-(1-iminoethyl)-lysine. Where the nitric oxide synthase inhibitor isa derivative of citrulline it may be, for example L-thiocitrulline,L-homothiocitrulline or an S-alkylthiocitrulline such asS-methyl-L-thiocitrulline. Where the nitric oxide synthase inhibitor isan aminopyridine it may be for example 2-amino-4-methylpyridine.

The composition is useful for the treatment of diseases involving activeangiogenesis for example solid tumours, psoriasis, diabetic retinopathy,macular degeneration, atherosclerosis and rheumatoid arthritis.

The relative proportion of each component will be determined by theidentity of each individual vascular damaging agent or nitric oxidesynthase inhibitor and by the disease to be treated.

The composition may include pharmaceutically acceptable excipientsselected with regard to the intended route of administration andstandard pharmaceutical practice. The composition may take a formsuitable for oral, buccal, nasal, topical, rectal or parenteraladministration and may be prepared in a conventional manner usingconventional excipients. For example for oral administration thecomposition may take the form of tablets or capsules. For nasaladministration or administration by inhalation the compounds may beconveniently delivered as a powder or in solution. Topicaladministration may be as an ointment or cream and rectal administrationmay be as a suppository. For parenteral injection (includingintravenous, subcutaneous, intramuscular, intravascular or infusion) thecomposition may take the form of, for example, a sterile solution,suspension or emulsion.

The dose of a compound of the invention required for the prophylaxis ortreatment of a particular condition will vary depending on the identityof the individual components, the route of administration, the form andseverity of the condition and whether the compound is to be administeredalone or in combination with another drug. Thus the precise dose will bedetermined by the administering physician and will depend on theparticular vascular damaging agent and NO synthase inhibitor in thecomposition. However the dose of the vascular damaging agent envisagedis, for example, in the range 10-1000 mg/m² body surface, preferably20-200 mg/m² and that for the nitric oxide inhibitor 1-1000 mg/m²,preferably 5-5000 mg/m². A unit dose form of the vascular damaging agentas, for example, a sterile solution for injection will usually contain,for example, 40-400 mg of the active ingredient. A unit dose form of thenitric oxide synthase inhibitor as, for example, a sterile solution forinjection will usually contain, for example, 10-1000 mg of the activeingredient. A unit dose form of a composition containing both a vasculardamaging agent and a nitric oxide synthase inhibitor as, for example, asterile solution for injection will usually contain, for example, 40-400mg of the vascular damaging agent and 10-1000 mg of the nitric oxidesynthase inhibitor.

The composition of the invention may be administered as a sole therapyor in combination with other treatments. For the treatment of solidtumours the composition may be administered in combination withradiotherapy or in combination with other anti-tumour substances forexample those selected from mitotic inhibitors, for example vinblastine,paclitaxel and docetaxel; alkylating agents, for example cisplatin,carboplatin and cyclophosphamide; antimetabolites, for example5-fluorouracil, cytosine arabinoside and hydroxyurea; intercalatingagents for example adriamycin and bleomycin; enzymes, for exampleaspariginase; topoisomerase inhibitors for example etoposide, topotecanand irinotecan; thymidylate synthase inhibitors for example raltitrexed;biological response modifiers for example interferon; antibodies forexample edrecolomab; and anti-hormones for example tamoxifen. Suchcombination treatment may involve simultaneous or sequential applicationof the individual components of the treatment.

In a further embodiment of the invention we provide the use of acompostion of the invention for the preparation of a medicament for thetreatment of a disease involving active angiogenesis.

The invention will now be illustrated by the following Examples in whichbiological assays are used to illustrate the invention:

Induction of Necrosis

Mice bearing either CaNT or SaS tumours were treated with the testcompound and tumours excised after 24 h, fixed in formalin, embedded inparaffin, sectioned and stained with haematoxylin and eosin. Sectionswere scored based on area of necrosis as follows: % necrosis score  0-101 11-20 2 21-30 3 31-40 4 41-50 5 51-60 6 61-70 7 71-80 8 81-90 9 91-100 10Control tumours had mean scores of 2.0 (CaNT) and 1.0 (SaS).

EXAMPLE 1

In this assay the effect of a given dose of either a vascular damagingagent or a nitric oxide synthase inhibitor administered alone can becompared with the effect of a combination of the two agents. TABLE 1Enhancement of Combretastatin A4 phosphate (CA4P) activity in SaStumours by coadministration of L-N^(G)-nitroarginine (L-NNA) Necrosisscore ± SEM Treatment (n) None 1.0 ± 0 (10) CA4P, 500 mg/kg 1.7 ± 0.7(3) L-NNA, 10 mg/kg 2.0 ± 1 (3) CA4P, 500 mg/kg + L-NNA 10 mg/kg 9.0 ± 0(3)

EXAMPLE 2

TABLE 2 Enhancement of Combretastatin A4 phosphate (CA4P) activity inSaS tumours by coadministration of 2-amino-4-methylpyridine (AMP)Necrosis score ± SEM Treatment (n) None 1.0 ± 0 (10) CA4P, 500 mg/kg 1.7± 0.7 (3) AMP, 10 mg/kg 1.0 (2) CA4P, 500 mg/kg + AMP 10 mg/kg 4.5 (2)

EXAMPLE 3 Activity Against Tumour Vasculature Measured by FluorescentDye

The following experiment further demonstrates the ability of thecompounds to damage tumour vasculature.

Tumour functional vascular volume in CaNT tumour-bearing mice wasmeasured using the fluorescent dye Hoechst 33342 according to the methodof Smith et al (Brit J Cancer 57, 247-253, 1988). The fluorescent dyewas dissolved in saline at 6.25 mg/ml and injected intravenously at 10mg/kg 24 hours after intra peritoneal drug treatment. One minute later,animals were killed and tumours excised and frozen; 10 μm sections werecut at 3 different levels and observed under UV illumination using anOlympus microscope equipped with epifluorescence. Blood vessels wereidentified by their fluorescent outlines and vascular volume wasquantified using a point scoring system based on that described byChalkley, (J Natl Cancer Inst, 4, 47-53, 1943). All estimates were basedon counting a minimum of 100 fields from sections cut at the 3 differentlevels. TABLE 3 Enhancement of Combretastatin A4 phosphate (CA4P)activity in CaNT tumours by coadministration of L-N^(G)-nitroarginine(L-NNA). Vascular Volume % ± SEM Treatment (n) None 2.35 CA4P, 25 mg/kg1.03 ± 0.14 (4) L-NNA, 10 mg/kg 2.45 ± 0.04 (3) CA4P, 25 mg/kg + L-NNA10 mg/kg 0.63 ± 0.25 (3)

1-15. (canceled)
 16. A pharmaceutical composition for the treatment of adisease involving active angiogenesis which comprises (a) a tubulinbinding agent in an amount sufficient to cause damage toneovaasculature, (b) an inhibitor of the formation of nitric oxide in amammalian system and (c) a pharmaceutically acceptable excipient.
 17. Apharmaceutical composition for the damage of the formation of newvasculature by angiogenesis comprising a combination of (a) a tubulinbinding agent in an amount sufficient to cause damage to neovasculature,(b) an inhibitor of nitric oxide synthase in an amount sufficient toaugment the effect of the tubulin binding agent and (c) apharmaceutically acceptable excipient.
 18. A composition according toclaim 2 wherein the inhibitor of nitric oxide synthase is selected froma derivative of arginine, ornithine, lysine, citrulline,S-alkylthioureas and aminoguanidine.
 19. A composition according toclaim 4 wherein the nitric oxide synthase inhibitor is anN^(G)-substituted L-arginine selected from N-nitro-L-arginine and alkylesters thereof, N^(G)-methyl-L-arginine and N^(G)-amino-L-arginine. 20.A composition according to claim 4 wherein the derivative of ornithineis L-N6-(1iminoethyl)-ornithine.
 21. A composition according to claim 4wherein the derivative of lysine is L-N6-(1-iminoethyl)-lysine.
 22. Acomposition according to claim 4 wherein the derivative of citrulline isselected from L-thiocitrulline, L-homothiocitrulline or anS-alkylthiocitrulline.
 23. A composition according to claim 1 whereinthe composition is in the form of a kit, one part of the kit containingthe tubulin binding agent and the second part of the kit the inhibitorof the formation of nitric oxide.